Method and reagents for genetic immunization

ABSTRACT

DNA vaccines which incorporate genetic sequences encoding sorting signals which direct an expressed antigen to a specific cellular organelle facilitate loading of the antigen onto a Class I or Class II MHC molecule for immune presentation. These vaccines are a nucleic acid construct of a genetic sequence encoding a protein or peptide antigen and a sorting signal which will direct expressed antigen to the ER or endosomal-lysosomal compartments within the cell. The resulting constructs can be used as naked DNA vaccines, packaged in liposomes, or coated onto colloidal gold particles. The construct might also be delivered in an expression vector which is expressed in cells of the organism being immunized.

[0001] This application relates to improved reagents for use in “geneticimmunization,” and to a method for genetic immunization which makes useof these reagents to elicit a more potent immune response.

[0002] The generation and regulation of immune response is a result of acomplex system of interactions between B- and T-lymphocytes, circulatingantibodies, and antigen presenting cells (APC). The induction of humoraland cell-mediated immune responses to protein antigens requires therecognition of the antigens by helper T (TH) cells. The reasons for thisis that helper T cell are necessary for stimulating B-lymphocyte growthand differentiation, and for activating the effector cells ofcell-mediated immunity, including macrophages and cytolytic Tlymphocytes (CTLs). Briefly, foreign antigen is processed by APCs whichresult in the generation of antigen-derived peptide fragments bound tothe major histocompatability complex (MHC) Class I and Class IImolecules (referred to as human leukocyte antigens or HLA Class I andClass II proteins in humans). These complexes which are found on thecell surface of the APC are then presented to TH cells. Recognition ofthe peptide-MHC complex by T cells is the initiating stimulus for T cellactivation. Thus, more efficient presentation of peptide-MHC complex canlead to more efficient T cell activation. Activation leads to thesecretion of cytokines, proliferation, and regulatory or cytolyticeffector functions which all lead to immunity, in part through theeradication of cells presenting antigen.

[0003] T cell-mediated eradication of cells expressing antigen can beaccomplished in three ways. First, humoral responses occur whenactivated TH cells stimulate the proliferation and differentiation ofspecific B cell clones to produce antibodies which eventually eliminatecells expressing the antigen as well as extracellular antigen. Second,cell-mediated responses occur when cytokines activate T cells todifferentiate into CTLs. The infected target cell is then lysed by theCTL. Endogenous antigens, such as viruses and tumor antigens, activateClass-I restricted CTLs, which lyse cells producing these intracellularantigens. Third, nonspecific responses occur when antigen-activated Tcells secrete cytokines that recruit and activate inflammatory cellssuch as macrophages and natural killer cells that are not specific forthe antigen. Overall, therefore, T cells play a central role inrecruiting a broad immune response.

[0004] As used herein, the term “genetic immunization” refers to the useof DNA as a vaccine to produce an immune response to the protein orpeptide antigen encoded by the DNA. Intramuscular administration ofnaked DNA has been shown to elicit both humoral and cellular immuneresponse. The precise mechanism by which DNA vaccines elicit an immuneresponse is not known, although several possibilities have beendiscussed. See Pardoll et al., “Exposing the Immunology of Naked DNAVaccines”, Immunity 3: 165-169 (1995). Regardless of the mechanism,however, the effectiveness of DNA vaccines to produce both humoral andcellular immunity indicates that naked DNA is expressed afteradministration, with the protein or peptide product being presented asan antigen in association with either Class I or Class II proteins.

[0005] The processing and presentation of antigens by Class I and ClassII molecules occurs in different organelles within the cells.Specifically, the endoplasmic reticulum (ER) has been shown to be thesite for loading peptide antigens derived from the cytoplasm onto ClassI molecules, while the endosomes/lysosomes have been shown to be thesite for loading peptide antigens onto Class II molecules. Thus, thetype of immune response and the extent to which an immune response isgenerated may depend in significant measure on the amount of antigenreaching the ER and endosomal loading sites. It would therefore behighly advantageous to be able to direct and control the accumulation ofantigen within a desired location within the cell to provide optimumimmune response.

[0006] It is an object of the present invention to control thetrafficking to and stability of selected antigens within specificcellular organelles, and to use this method to provide for enhancedgenetic immunization.

[0007] It is a further object of the present invention to provide DNAvaccines which incorporate genetic sequences encoding sorting signalswhich direct the expressed antigen to a specific cellular organelle andfacilitate loading of the antigen onto a Class I or Class II MHCmolecule for immune presentation.

[0008] It is still a further object of the invention to provide a methodfor genetic immunization utilizing DNA vaccines which incorporategenetic sequences encoding sorting signals which direct the expressedantigen to a specific cellular organelle and facilitate loading of theantigen onto a Class I or Class II MHC molecule for immune presentation.

SUMMARY OF THE INVENTION

[0009] These and other objects of the invention are achieved through theconstruction of a genetic sequence encoding a protein or peptide antigenand a sorting signal which will direct expressed antigen to the ER orendosomal-lysosomal compartments within the cell. The resultingconstructs are useful as DNA vaccines, and can be used as naked DNA,packaged in liposomes, or coated onto colloidal gold particles. Theconstruct might also be delivered in an expression vector, for example aviral vector, which is expressed in cells of the organism beingimmunized.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 shows three forms of an ovalbumin/gp75 fusion protein;

[0011]FIG. 2 shows the induction of CD4+ T cell response using theinvention;

[0012]FIG. 3 shows the induction of an IgG response using the invention;

[0013]FIG. 4 shows the affect of genetic immunization in accordance withthe invention on tumor growth in mice;

[0014]FIG. 5 shows a method for forming a nucleic acid construct inaccordance with the invention;

[0015]FIG. 6 shows the construction of three different plasmidscontaining a construct in accordance with the invention; and

[0016]FIG. 7 shows a graphical representation of the immune responsegenerated upon immunization with a construct in accordance with theinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0017] In accordance with the present invention, nucleic acid constructsfor use in genetic immunization procedures are prepared which comprise

[0018] (a) an antigen-coding region encoding an antigenic protein orpeptide; and

[0019] (b) a sorting region encoding a protein or peptide which acts asa sorting signal to direct intracellular transport of the protein orpeptide to the endosomal-lysosomal compartments or their transportto/retention in the endoplasmic reticulum of a cell. As used herein, theterm “nucleic acid construct” reflects the fact that the material isproduced from component parts that are spliced together from differentsources and excludes, for example, a DNA molecule encoding a naturallyoccurring protein that includes both an antigenic determinant and asorting signal region.

[0020] The antigen-coding region of the nucleic acid polymers of theinvention is selected to encode for one or more desired antigenicdeterminants of a protein or peptide of interest. Thus, theantigen-coding region may encode an entire protein or peptide, or animmunogenic portion thereof associated with a selected epitope of theprotein or peptide.

[0021] The sorting region employed in the nucleic acids polymers of theinvention is selected to provide a peptide region that directsintracellular transport of an expressed protein or peptide to a desiredintracellular location. Suitable sorting signals for directingintracellular transport of the expressed antigen to the endosomesinclude the following molecules: the signal from human gp75 (brown locusprotein) which includes the signal region

[0022] Glu Ala Asn Gln Pro Leu Leu Thr Asp; SEQ ID No. 1

[0023] the signal from human tyrosinase (albino locus protein) whichincludes the signal region

[0024] Glu Glu Lys Gln Pro Leu Leu Met Asp; SEQ ID No. 2

[0025] the signal from human gp100 (silver locus protein, Pmel 17) whichincludes the signal region

[0026] Glu Asp Ser Pro Leu Leu; and SEQ ID No. 3

[0027] the signal from human P-protein (pink eyed locus) which includesthe signal region

[0028] Glu Asp Thr Pro Leu Leu SEQ ID No. 4

[0029] as described in Vijayasaradhi et al., “Intracellular Sorting andTargeting of Melanosomal Membrane Proteins: Identification of Signalsfor Sorting of the Human Brown Locus Protein, GP75” J. Cell Biology 130:807-820 (1995).

[0030] Suitable sorting signals for directing intracellular transport ofthe expressed antigen to the endoplasmic reticulum (or retentiontherein) include the signal region

[0031] Pro Ser Arg Asp Arg Ser Arg His Asp Lys Ile His SEQ ID No. 5

[0032] which has been shown to retain a viral glycoprotein in theendoplasmic reticulum. Rose et al. “Altered cytoplasmic domains affectintracellular transport of the vesicular stomatitis virus glycoprotein”Cell 34: 513 (1993); Bartido et al. “Processing of a viral glycoproteinin the endoplasmic reticulum for class II presentation” Euro. J Immunol.25: 22111-2219 (1995).

[0033] Mutant forms of naturally-occurring sorting signal-containingproteins and peptides may also be used as the sorting region of theinvention. Such mutants can alter protein or peptide trafficking bymaking the protein or peptide more unstable in a particular compartmentin the cell, including the ER and endosome/lysosome. For example,because glycosylation plays an important role in stabilizing endocyticmembrane proteins within different cellular compartments, glycosylationmutants can be used to more closely control the intracellular transportof the expressed antigen/sorting signal product to induce the desiredform of immune response. On any given protein, there will generally bemultiple glycosylation sites, with each site being of differentimportance in its effect on the transport and degradation of theprotein. For example, in the case of mouse gp75, there are fiveN-glycosylation sites, one of which strongly effects the resistance toprotease digestion and two others of which are important for permittingexport of the protein from the endoplasmic reticulum. Other mutantsforms, for example mutants forms which disrupt the sorting signalregions described above, particularly the Pro Leu Leu motif, can also beused in the method of the invention. Constructs which have a sequencewhich is the same as the wild-type sequence for a sorting signal, orwhich are a mutant variant of such a wild-type, or which aresynthetically generated to encode the same protein/peptide sequence asthe wild-type or mutant variants based upon degeneracy of the nucleicacid code are referred to in the specification and claims hereof asbeing “derived from” the wild type protein or peptide.

[0034] Identification of suitable mutants can be identified by creationof the desired mutant, for example by site-directed mutagenesis,followed by testing of each mutant in a model system. For example, inthe case of gp75, the two mutant forms shown in FIG. 1 were compared tothe wild-type mutant. The sorting signal peptide labeled as “Deletion”has a deletion mutation introduced in the region spanning Asn 511 to Asp517, which results in the deletion of the sorting signal region. Thesorting signal peptide labeled L2A differs from the wild-type by asingle base substitution, Leu514 to Ala514 which disrupts the Pro LeuLeu motif of the sorting signal.

[0035] Testing of the ova/gp75 constructs shown in FIG. 1 for theirability to induce an immune response to ova in mice showed differentialresults depending on the sorting signal employed. As shown in FIGS. 2and 3, the construct containing the wild-type sequence produced a MUCHgreater CD4⁺ T Cell response, while the constructs containing the mutantsequence produced higher levels of IgG response. Both the fusion proteincontaining the wild-type sequence and the fusion protein containing theL2A mutation were effective to provide protection against tumor cellsexpressing ovalbumin. (FIG. 4).

[0036] Glycosylation-mutants of sorting signals can be similarlyprepared and tested for their ability to direct the trafficking of theexpressed fusion proteins to desired regions within the cell. Mutationsat two of the five identified glycosylation sites on mouse gp75 (Asn304and Asn385) produced proteins which are apparently retained and degradedin the endoplasmic reticulum. Such mutant sorting signals can be used infusion proteins in accordance with the invention to selectively generatepeptides for the MHC Class I pathway. A third mutant form (Asn350) wastransported from the ER to the Golgi apparatus at a similar rate to thewild-type but exhibited a markedly decreased half-life, being veryunstable in endosomes. Such mutants can be used in fusion proteins todirect an MHC Class II response, and the enhanced degradation of thesorting signal may generate more peptides for presentation through thispathway. Mutation at Asn181 of mouse gp75 impacted the rate of transportwith the result that the mutant protein tended to localize in theendosomal/lysosomal structures of the transfectants and not in the Golgiapparatus. Fusion proteins with sorting signals of this type can also beused to direct an MHC Class II response to the antigen.

[0037] The antigen-coding region and the sorting region are combinedinto a single nucleic acid polymer which may optionally contain a linkerregion to ensure proper folding of the encoded fusion protein. Onesuitable technique for this process utilizes initial separate PCRamplification reactions to produce the two regions, each with a linkersegment attached to one end, followed by fusion of the two amplifiedproducts in a further PCR step using the general scheme shown in FIG. 5.This technique is referred to as linker tailing. Of course, it will beappreciated that other techniques and variations on this technique canbe used. For example, when either the antigen-coding region or thesorting region is fairly short, the region may be chemically synthesizedand coupled to the other region by ligation. Suitable restriction sitesmay also be engineered into regions of interest, after which restrictiondigestion and ligation is used to produce the desired fusion-proteinencoding sequence.

[0038] After synthesis, the nucleic acid polymer containing both theantigen-coding region and the sorting region is combined with a promoterwhich is effective for expression of the nucleic acid polymer inmammalian cells. This can be accomplished by digesting the nucleic acidpolymer with a restriction endonuclease and cloning into a plasmidcontaining a promoter such as the SV40 promoter, the cytomegalovirus(CMV) promoter or the Rous sarcoma virus (RSV) promoter. The resultingconstruct is then used as a vaccine for genetic immunization. Thenucleic acid polymer could also be cloned into plasmid and viral vectorsthat are known to transduce mammalian cells. These vectors includeretroviral vectors, adenovirus vectors, vaccinia virus vectors, poxvirus vectors and adenovirus-associated vectors.

[0039] The nucleic acid constructs containing the promoter,antigen-coding region and sorting region can be administered directly orthey can be packaged in liposomes or coated onto colloidal goldparticles prior to administration. Techniques for packaging DNA vaccinesinto liposomes are known in the art, for example from Murray, ed. “GeneTransfer and Expression Protocols” Humana Pres, Clifton, N.J. (1991).Similarly, techniques for coating naked DNA onto gold particles aretaught in Yang, “Gene transfer into mammalian somatic cells in vivo”,Crit. Rev. Biotech. 12: 335-356 (1992), and techniques for expression ofproteins using viral vectors are found in Adolph, K. ed. “Viral GenomeMethods” CRC Press, Florida (1996).

[0040] The compositions of the invention are preferably administeredintradermally, subcutaneously or intramuscularly by injection or by gasdriven particle bombardment, and are delivered in an amount effective toproduce an immune response in the host organism. The compositions mayalso be administered ex vivo to blood or bone marrow-derived cells(which include APCs) using liposomal transfection, particle bombardmentor viral infection (including co-cultivation techniques). The treatedcells are then reintroduced back into the mammal to be immunized. Whileit will be understood that the amount of material needed will depend onthe immunogenicity of each individual construct and cannot be predicteda priori, the process of determining the appropriate dosage for anygiven construct is straightforward. Specifically, a series of dosages ofincreasing size, starting at about 0.1 ug is administered and theresulting immune response is observed, for example by measuring antibodytiter using an ELISA assay, detecting CTL response using a chromiumrelease assay or detecting TH response using a cytokine release assay.

[0041] The invention will now be further described and illustrated bywas of the following, non-limiting examples.

EXAMPLE 1

[0042] To demonstrate the creation of a nucleic acid construct inaccordance with the invention, a construct having an antigen-codingregion encoding chicken ovalbumin and a sorting region derived frommurine gp75 was produced. In the construct, the chimeric protein of fulllength ovalbumin and the C-terminal region of gp75 containing thesorting sequence

[0043] Glu Ala Asn Pro Leu Leu Thr Asp SEQ ID No. 1

[0044] are connected with a nine amino acid linker,

[0045] Ser Gly Gly Ser Gly Gly Ser Gly Gly. SEQ ID No. 6

[0046] The construct was prepared using a series of PCR reactions.First, the ovalbumin gene coding amino acids 1-386 was amplified frompAc-neo-OVA (Moore et al., “Introduction of Soluble Protein into theClass I Pathway of Antigen Processing and Presentation” Cell 54: 777-785(1988)) with the primer pair

[0047] 5′-CGCCACCAGACATAATAGC-3′ and SEQ ID No. 7

[0048] 5′-GCCTCCTGAACCTCCGGAACCACCAGAAGGGGAAACACATCTGCC-3′. SEQ ID NO. 8

[0049] The transmembrane and cytoplasmic domains of gp75, amino acid488-539, were then amplified out from pSVK3-mpg75 (Vijayasaradhi et al.,J. Cell Biol. 130: 807-820 (1995) using primers

[0050] 5′-TCTGGTGGTTCCGGAGGTTCAGGAGGCATCATTACCATTGCTGTAGTG-3′ SEQ ID No.9

[0051] and 5′-GGTTGCTTCGGTACCTGCTGCG-3′. SEQ ID No. 10

[0052] The PCR products from these two amplification were purified andsubjected to a second round of PCR using primers5′-CGCCACCAGACATAATAGC-3′ (SEQ ID NO. 11) and5′-GGTTGCTTCGGTACCTGCTGCG-3′ (SEQ ID No. 12). (See FIG. 5) The secondphase of the PCR fused the ova and gp75 sorting region with the designedlinker in between. Thus, the construct has a combined open reading frameof 1365 base pairs capable of coding a protein a protein of 455 aminoacids which includes 386 amino acids from ovalbumin, 9 amino acids fromthe linker and 60 amino acids from gp75.

[0053] The construct was digested with EcoR1 and Kpn1 and cloned intopSVK3 (Pharmacia/LKB Ltd.), pBK-CMV and pBK-RSV (Stratagene Inc.)separately as shown in FIG. 6. These constructs have been sequenced andtheir structures have been confirmed.

[0054] To construct targeting derivative mutants, PCR primers containingmutations were synthesized. They are5′-CTCAGCATAGCGTTGATAGTGATTCTTGGTGCTTCTAGAACG-3′ SEQ ID No 13 and5′-CGTTCTAGAAGCACCAAGAATCACTATCAACGCTATGCTGAG-3′ (SEQ ID No. 14) for thedeletion of Asn511 to Asp517 mutant. The primer pair5′-GAGTGCAGGCTGGTTGGCTTC-3′ (SEQ ID No. 15) and5′-CCTGCACTCACTGATCACTAT3′ (SEQ ID NO. 16) are used to construct theLeu514 to Ala514 mutant (FIG. 1). These constructs have been sequencedand their structures have been confirmed.

EXAMPLE 2

[0055] Expression of the fusion protein as well as that of ovalbuminalone was examined by utilizing plasmids that contained the encoding DNAunder different promoters. The DNA was transiently transfected intomouse L cells or monkey COS cells by calcium phosphate precipitation orDEAE-chloroquine methods. The cells (1×10⁵) were plated on a 8-wellchamber slide (Nunc, Inc.) and incubated for 24 hours. Cells were thentransfected with 0.5-1.0 μg DNA by known standard calcium phosphate orDEAE methods. After transfection, cells were allowed to grow for 24-48hours prior to determining the intracellular localization of ovalbuminin the transfected cell.

[0056] Detection of the protein was carried out by immunofluorescentstaining of the antigen. Cells were washed with cold phosphate bufferedsaline (PBS), fixed with 2% formaldehyde, permeabilized with methanol at−20° C. and then incubated with the monoclonal antibody (mAb) OVA-14(BioMaker Inc.). The cellular localization of the antigen was thenvisualized by staining with a secondary antibody, FITC-conjugated goatanti-mouse antibody (Dako, Inc.). The cells were observed using a Nikonmicroscope and photographed using back and white film, ISO100.

[0057] Expression of the protein ovalbumin or of the fusion proteingp75-ova was observed upon transfection of the DNA constructs into cellsusing all three promoters (SV40, CMV and RSV). Furthermore, whenplasmids which included gp75 sorting region were introduced into thecells, localized vesicular immunofluorescent staining was observedconsistent with endosome-lysosome localization. In contrast, when thecontrol plasmids without the gp75 sorting region were introduced, a morediffuse cytoplasmic staining pattern typical of staining of Golgi/ERlocalization was observed. Thus, incorporation of the gp75 sortingregion dramatically changed the intracellular trafficking of a protein,ovalbumin, destined for the secretory pathway to a protein contained ina vesicular compartment in the endocytic pathway.

EXAMPLE 3

[0058] To test the ability of the constructs encoding the OVA/gp75fusion protein to act as a vaccine for genetic immunization,(C57BL/6xBalb/c) F1 mice were immunized with respective DNA plasmidspurified by using QIAGEN ion-exchange columns (Qiagen, Inc.). DNA (100μg in 100 μl of a 25% sucrose solution in PBS) was injectedsubcutaneously at day 0 and day 14. Blood samples were collected at day14 and day 28.

[0059] Antibody response was monitored using an ELISA assay. Chickenovalbumin (Sigma, Inc.) was used as the antigen and plated in a 96-wellplate overnight at 4° C. The diluted serum samples were then added tothe plate and incubated for 1 hour at room temperature. After washing,the secondary antibody, alkaline phosphatase-conjugated goat anti-mouseIgG, was added and plate was incubated for 1 hour. Color development wasachieved upon addition of the Sigma Fast p-nitrophenyl substrate.Reaction was terminated with the addition of 3N NaOH. Absorbance in thedifferent wells was obtained using the BioRad EIA Reader 2550. Resultsare shown in FIG.7. As can be seen, the genetic immunization techniqueusing the constructs of the invention was effective to produceantibodies to ovalbumin. Corresponding plasmid constructs with differentpromoters also elicited an immune response, although not as strongly asthat seen when using the SV40 promoter.

EXAMPLE 4

[0060] CBF1 mice were immunized with DNA plasmids purified by the QIAGENion-exchange columns (Qiagen, Inc.). To prepare bullets forimmunization, 50 mg of 0.95-2.6 μm gold particles (Auragen, Inc.) weremixed with 0.05-0.1 M spermidine, 100 μg of plasmid DNA was added to themixture, and 1.0-2.5 M CaCl₂ was added dropwise while vortexing. Afterprecipitation, the gold/plasmid DNA complex was washed three times withcold 100% ethanol. Seven ml of ethanol was added to the pellet toachieve a bead loading rate of 0.5 mg gold and 1.0 μg plasmid DNA perinjection. The gold/plasmid DNA solution was then instilled into plasticTefzel® tubing, the ethanol gently drawn off, and the tube purged withnitrogen gas at 400 ml/min for drying. The tube was cut into 0.5 inchbullets and these were used for immunization. For cutaneousimmunizations, all mice were anesthetized with Metofane inhalation(Pitman-Moore, Mundelein, Ill.). Abdominal hair was removed with Nair®depilatory cream (Carter-Wallace, New York, N.Y.), so that depilatedabdominal skin was exposed for immunization. The bullets were placedinto a hand-held helium-driven gene gun (Auragen, Inc.). Animals wereimmunized by delivering the gold beads in one bullet into each abdominalquadrant, for a total of four injections per immunization. Eachinjection delivered 1 μg DNA and therefore a total of 4 μg DNA per mouseeach immunization. Each bullet was delivered to the abdominal skin at ahelium pressure of 400 pounds per square inch.

[0061] In vivo antibody response. Indirect ELISA assays were performedto monitor the antibody response. CB6F1 mice were immunized withdifferent plasmid constructs by gene gun once a week for four weeks anda boost at week 6. Serum samples were collected at weekly intervals.Purified chicken ovalbumin (Sigma, Inc.) was used as the antigen andplated 50 μg each well in a 96-well plate overnight at 4° C. The dilutedserum samples were then added to the plate and incubated for 1 hour atroom temperature. After washing, the second antibody goat anti-mouse IgGconjugated with alkaline phosphatase (Sigma, Inc.) was added andincubated for 1 hour at 37° C. The plates were developed using the Fastp-Nitrophenyl phosphate substrate (Sigma, Inc.) and the reactions wereterminated with the addition of 3N NaOH. The absorbance at 605 nm wereobtained by the BioRad EIA Reader 2550 (BioRad Inc.). The positivecontrol group immunized with naked DNA containing the full lengthovalbumin generated strong response within 2 weeks (FIG. 3). Mutantswith a disrupted (L2A) or deleted (del) sorting signal also generatedantibody response although the response appeared to be delayed comparingto the wild type ovalbumin. Interestingly, the ova/gp75 fusion proteinfailed to generate an antibody response under the particularimmunization protocol. The reason for that is not known. But it isconceivable that most of the fusion protein is sequestered in the celldue to its retention signal at the c-terminus and not efficientlyrecognized by the B cells.

[0062] CD4+ T cell proliferative assay. A proliferation assay wascarried out to monitor the efficiency of in vivo priming of CD4+ T cellsby the different DNA constructs. CB6F1 mice were immunized once a weekfor two weeks by gene gun and at day 14 the mice were sacrificed. CD4+ Tcells were purified from pooled splenocytes using a CELLECT™.PLUS column(Biotex Laboratories, Inc.). The purified CD4+ T cells (3×10⁵) were invitro stimulated by incubation with syngeneic naive splenocytes (1×10⁵)pulsed with the denatured ovalbumin at different concentrations for 4days at 37° C. On day 4, 100 μCi of ³H-TdR was added to each well andthe cpm is counted after 16-18 hours. The proliferation response isexpressed as the net cpm subtracting the background (FIG. 2). Theova/gp75 fusion efficiently primed T cells in vivo suggesting theendogenous processing and presentation of the fusion protein. Moreover,melanosomal targeting mutants, L2A and Del, did not prime welldemonstrating the requirement of the targeting signal for the functionof the fusion protein in stimulating CD4+ T cell prliferation.

[0063] Tumor protection. CB6F1 mice were immunized weekly for two weekswith pBK-CMV vector alone, the vector contaning the ova-gp75 constructof FIG. 1 or the vector contaning the ova/L2A construct. On day 14, theimmunized mice were challenged by injecting subcutaneously with 1×10⁶MO4 melanoma cells, a B16 melanoma cell line transfected with the fulllength ovalbumin. Mice were checked for tumor growth every other dayover a period of 3 weeks. The “no treatment” group (n=10) all developedpalpable tumor within 2 weeks. Similarly, of the mice immunized with thevector alone (n=10), all but one developed tumor. None of the miceimmunized with the vecotr encoding the ova/gp75 fusion protein (n=10)developed tumor and only one out of ten developed tumor in the L2A group(FIG. 4). This result clearly shows that the immune response elicited byimmunization with fusion protein construct can lead to protection oftumor challenges in vivo.

[0064] To test the whether immunization by this method inducedimmunologic memory, mice immunized with the fusion DNA werere-challenged with tumor MO4 or B16 melanoma (the parent melanoma cellline of MO4 that does not express the antigen ovalbumin) five weeksafter the last immunization. None of the five mice challenged with MO4developed tumor when observed for at least six weeks, and two of thefive mice challenged with B16 parental melanoma did not develop tumorover at least 6 weeks. All five unimmunized mice challenged with B16developed tumors within 10-14 days. The sorting signal was required forprotection against B16 parental tumor. Five mice immunized with the DNAconstruct containing a mutant sorting signal (L2A) all developed tumorwhen challenged with B16. The sorting signal was also required forpotent immunological memory, because one out of four mice immunized withconstruct containing mutant sorting signal (L2A) developed tumor withMO4 tumor challenge. This experiment show that immunization with DNAconstructs containing the tyrosinase family sorting signal can providelong lasting memory against the antigen and can even provide a broaderprotection against tumor challenge in tumors that do not express theantigen.

EXAMPLE 5

[0065] To identify the sorting signal, constructs were made having anantigen-coding region encoding the extracellular domain of the Tlymphocyte surface glycoprotein CD8, and a sorting region containing thecytoplasmic tail of the human gp75 (amino acids 497 to 537) or thecytoplasmic tail and the transmembrane domains (TM) of human gp75 (aminoacids 477 to 537). To make these constructs, a full-length 2.8 kb EcoRIfragment was isolated from a human melanoma cDNA library and subclonedinto the unique EcoRI site of eukaryotic expression vectors pCEXV3(Bouchard et al., J. Exp. Med. 169: 2029-2042 (1989)) or pSVK3.1 (aderivative of vector pSVK3 obtained by deletion of the Sac I fragmentwithin the multiple cloning site), or SmaI site of pSVK3 (Pharmacia LKB,Piscataway, N.J.) following a fill-in reaction with Klenow fragment ofDNA polymerase (New England Biolabs, Beverly, Mass.). The orientation ofthe cloned insert was determined by restriction analysis and confirmedby dideoxy chain termination sequencing method (Sequenase Kit, USBiochemicals, Cleveland, Ohio) using an oligonucleotide primercomplementary to the vector sequences upstream of the cloning site.

[0066] Mouse L cell fibroblasts were transfected with plasmid containinggp75 cDNA and pSV2neo. Transfected clones were isolated by selecting forgrowth in the antibiotic G418 (1 mg/ml; Gibco BRL, Gaithersburg, Md.),and screened for gp75 expression by immuno-fluorescence staining withthe mAb TA99 (Vijayasaradhi et al., Exp. Cell Res. 171: 1375-1380(1991)).

[0067] The plasmid EBO-pCD-Leu2 containing human CD8α cDNA was obtainedfrom American Type Culture Collection (Margolskee et al., 1988). The 2.3kb BamHI fragment from this plasmid was isolated, made blunt-ended withKlenow fragment and cloned into the SmaI site of the expression vectorpSVK3. The orientation of the cDNA insert in the recombinant plasmids inE. coli DH5α was analyzed by appropriate restriction enzyme digestions,and confirmed by DNA sequencing.

[0068] Chimeric cDNAs encoding fusion proteins CD8/gp75(TM+Cyt) andCD8/gp75(Cyt) were constructed by the following methods. First,appropriate restriction sites at or near the TM/Cyt junction of CD8, andlumenal/TM and TM/Cyt junctions of gp75 were generated by site-directedmutagenesis (Kunkel et al., 1987) using Mutagene kit (BioRadLaboratories, Hercules, Calif.). Specifically, a mutant gp75 plasmidpSVgp75RV was generated by introducing an EcoRV restriction site atnucleotide 1560 (lumenal/TM junction) of gp75 cDNA in plasmid pSVK3using the mutagenic oligonucleotide

[0069] 5′-TACTGCTATGGCAATGA TATCAGGTACACTA-3 SEQ ID No. 17

[0070] (mutations introduced are shown in bold and underlined). Thisresulted in the conversion of glutamic acid at position 477 (amino acidsnumbered starting with the methionine coded by the initiation codon) toaspartic acid. Mutant plasmids pSVgp75H and pSVleu2H were generated byintroducing a HindIII restriction site in gp75 cDNA at nucleotide 1627,(gp75 TM/Cyt junction) and at nucleotide 706 (CD8 TM/Cyt junction) inCD8 cDNA using the mutagenic oligonucleotides

[0071] 5′-GCGTCTGGCACGAAGCTTATAAGAAGCAGT-3′ and SEQ ID No. 18

[0072] 5′-GTCTTCGGTTCCTAAGCTTGCAGTAAAGGGT-3′, SEQ ID No. 19

[0073] respectively. This resulted in conversion of leucine at position500 to lysine and isoleucine at 501 to leucine in gp75; and asparagineat 207 to lysine and histidine at 208 to proline in CD8. Mutants werefirst identified by appropriate restriction enzyme digestion andconfirmed by sequencing the relevant regions of the plasmids using aSequenase sequencing kit. Transient expression in mouse fibroblasts andimmunofluorescence analysis with mAbs TA99 (anti-gp75) and OKT-8(anti-human CD8) showed that intracellular staining of mutant proteinswas identical to the distribution of wild type counterparts, I. e.,punctate cytoplasmic staining of gp75 and cell surface expression ofCD8.

[0074] Plasmid pSVgp75RV was digested with EcoRV and XbaI to produce a≈1.2 kb fragment containing the TM+Cyt sequence and 3′ untranslatedsequence of gp75 cDNA including part of the multiple cloning sitesequences of the vector; plasmid pSVleu2H was digested with EcoRV andXbaI and the large ≈4 kb plasmid DNA fragment lacking TM and Cytsequences of CD8 cDNA was isolated. The 1.2 kb EcoRV-XbaI gp75 fragmentwas ligated with the large EcoRV-XbaI pSVleu2H fragment to generate aplasmid construct encoding the fusion protein CD8/gp75 (TM+Cyt).Similarly, a ≈1 kb HindIII-XbaI gp75 cDNA fragment (containing gp75 Cytand 3′ untranslated sequences), and a ≈4 kb HindIII-XbaI CD8 cDNAplasmid fragment (lacking the cytoplasmic tail sequences of CD8) wereisolated, respectively, from plasmids pSVgp75H and pSVleu2H, and ligatedto generate the fusion protein CD8/gp75(Cyt). Regions of the plasmids atthe CD8/gp75 junctions were sequenced from at least two independentclones to confirm the restoration of the reading frame. Large scaleplasmid preparations (Quiagen, Inc., Chatsworth, Calif.) were furtherverified by restriction enzyme digestions for the presence of enzymesites unique to gp75 and CD8 at appropriate regions in the chimericplasmids.

[0075] pSVK3.1gp75, was utilized to generate carboxyl terminal deletionmutants. The restriction enzyme site BglII at nucleotide 2000 of gp75cDNA is a unique site within the plasmid pSVK3.1. Plasmid pSVK3.1gp75(10 μg) was linearized by digestion with 40 units of BglII in a 50 μlreaction for 3 h at 37° C. Linearized ≈6.7 kb DNA was then digested for3-4 min with Bal 31 nuclease (1 unit enzyme/μg DNA) in 50 μl reaction.Digested DNA was immediately extracted with phenol:chloroform toinactivate and remove the nuclease, and the ends were filled in byKlenow fragment of DNA polymerase I to increase the population ofblunt-ended molecules (Sambrook et al., 1989). Klenow fragment wasinactivated by heating at 75° C. for 10 min, and a suppressible readingframe termination linker containing restriction site NheI,5′-CTAGCTAGCTAG-3′ (Pharmacia), was ligated to the blunt-ended,truncated pSVK3.1gp75 DNA molecules with 1 unit of T4 DNA ligase in 20μl reaction for 3 h at room temperature. The ligation mixture was usedto transform E. coli strain DH5α. Ampicillin-resistant bacterialcolonies were analyzed by agarose gel slot lysis method for the presenceof plasmid DNA of appropriate size. Plasmid DNA from 15 transformantswas isolated, analyzed by restriction enzyme digestion, and partiallysequenced to determine the number of bases deleted from the carboxylterminus and to confirm the addition of termination linker.

[0076] A transient transfection method was developed and optimized tostudy the intracellular distribution of gp75 expressed by mutantconstructs. Briefly, 2-4×10⁴ SK-MEL-23 clone 22a melanoma cells andmouse L cells fibroblasts were plated in 8-well LabTek chamber slides.The cells were transfected with plasmid DNA by calcium phosphateprecipitate method for 16-24 h, and then allowed to accumulate theexpressed protein for 12 to 48 h which was evaluated byimmunofluorescence microscopy and immunoelectronmicroscopy.

[0077] For immunofluorescence microscopy, cells on the 8-well glassslides were fixed with formaldehyde, followed by methanol, and stainedwith gp75 specific mouse mAb TA99 or OKT-8 followed by FITC-conjugatedanti-mouse IgG. Cells were examined under Nikon Optiphot fluorescencemicroscope and photographed using Kodak Ektachrome film.

[0078] For immunoelectronmicroscopy, mAb TA99 directly conjugated to 10nm gold particles was used for localization of gp75 by immunoelectronmicroscopy. Colloidal gold was prepared as described (Smit and Todd,1986) and mAb TA99-gold conjugate was prepared according to Alexander etal., 1985. Human melanoma SK-MEL-19 cells were fixed with 0.2%glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, infused with 2.3 Msucrose in PBS and the cell pellet was then frozen in liquid nitrogen.Ultrathin sections were cut and collected on formvar-carbon coatednickel grids. The sections on the grids were incubated in 0.5% BSA inPBS to block nonspecific protein binding sites and then stained with mAbTA99 conjugated to 10 nm gold particles. Washing and staining of thesections was performed according to Griffiths et al., 1983. Sectionswere observed on a Jeol 100CX electron microscope.

[0079] These experiments showed that the expressed proteins fromconstructs having a sorting region that included the 36 amino acidcytoplasmic tail of human gp75 (with or without the transmembraneregion) were localized to the juxtanuclear region of the cells, andthere was little or no staining of other cytoplasmic structures of theplasma membrane. This pattern showed localization of the expressedprotein in the Golgi region and possible other organelles such as lateendosomes and lysosomes present in the Golgi region. It was furtherdetermined, however, that the absence of cell surface staining whichwould be expected because of the presence of the CD8 portion of thechimeric protein is probably the result of protease degradation of theCD8 within the protease-rich endosomes and lysosomes.

EXAMPLE 6

[0080] The role of specific N-glycans in determining stability of anendocytic membrane protein within different cellular compartments wasinvestigated. The tyrosinase family of glycoproteins has multipleconserved potential N-linked glycosylation sites. The mouse brown locusprotein, gp75, is a prototype of the TRP family. We examined howN-linked glycosylation on gp75 plays a role in maintaining the stabilityof this protein as it is transported through different compartments, bysystemically eliminating each N-linked glycosylation sites.

[0081] An 1.8 Kb EcoR I fragment containing the full length mouse gp75cDNA was isolated from pMT4 plasmid (kindly provided by Dr. T.Shibahara, Tohoku University School of Medicine, Japan), and subclonedinto the unique EcoR I site of eukaryotic expression vector pSVK3.1 togenerate pSVK3.1-mgp75. pSVK3.1 is a derivative of pSVK3 (Pharmacia LKBBiotechnology, Inc., Piscataway, N.J.), modified by removing the Sac Ifragment within the multiple cloning sites. The orientation of theinsert was determined by restriction enzyme analysis and confirmed byDNA sequencing using Sequenase Kit (US Biochemicals, Cleveland, Ohio).The Muta-gene Phagemid in vitro mutagenesis kit (Bio-Rad, Melvile, N.Y.)was used to create Asn to Gln mutations at amino acid positions 96, 104,181, 304, 350 and 385, using the following oligonucleotidesrespectively.

[0082] OLXU 27: 5′-CTGACATGTTCTCTGAAAGAACCTCAGAGG-3′; SEQ ID NO. 21

[0083] OLXU 28: 5′-GTGTCCTGAGAACTGATCATTGCACTGACA-3′; SEQ ID NO. 22

[0084] OLXU 29: 5′-ATAAACGGAAATCTGCTCAAATTGTGGTGT-3′; SEQ ID NO. 23

[0085] OLXU 30: 5′-ACCCTCAGTGCTCTGACAAAGTGTTCCCAG-3′; SEQ ID NO. 24

[0086] OLXU 31: 5′-ACTGTCTGTAGACTGGGAATAAAAAGGAGG-3′; SEQ ID NO. 25

[0087] OLXU32: 5′-TCCTCCCGTTCCCTGCAGGAAGAGGTG-3′. SEQ ID NO. 26

[0088] Mutagenesis with above mutagenic primers resulted in conversionsof Asn (AAC or AAT) to Gln (CAG) at respective sites. The resultingmutant constructs were designated gp75g1, gp75g2, gp75g3, gp75g4, gp75g5and gp75g6. Mutants were screened and identified by DNA sequencing.

[0089] Mouse L cell fibroblasts were transfected with plasmidscontaining full-length or mutant gp75 cDNA and pSV2 neo using calciumphosphate precipitation method. The transfectants were selected forgrowth in medium containing 500 μg/ml effective concentration ofantibiotic Geneticin (GIBCO BRL Life Technologies, Grand Island, N.Y.).Individual transfectant clones were isolated using cloning rings(Bellco, Vineland, N.J.) and screened for gp75 expression byimmunofluorescence staining with mAb TA99.

[0090] We first investigated which potential N-linked glycosylationsites were used by comparing the molecular mass difference of mutantgp75 proteins to immature, glycosylated wild-type gp75. Transfectantsexpressing different gp75 glycosylation mutants were labeled with [³⁵S]methionine for 15 min followed by immunoprecipitation with mAb TA99. B16melanoma cells and wild-type gp75 transfectants produced a sharp 71 kDaband of gp75, representing an immature form of gp75 with high mannosesugar chains characteristic of ER processing. Among the glycosylationmutants, only gp75g2 appeared as a 71 kDa band, while all othersproduced a 68 kDa band. Because one high mannose oligosaccharide chaincorresponds to approximately 3 kDa of molecular mass, the observeddifference between the molecular mass of the mutant gp75 proteins andwild-type gp75 is consistent with the interpretation that the 68 kDamutant gp75 molecules contained one less carbohydrate chain than thewild-type gp75. This, in turn, is a direct result of the abolishment ofone carbohydrate chain at the particular potential glycosylation site.Thus, it is reasoned that these sites (Asn positions 96, 181, 304, 350and 385 which are individually mutated in gp75g1, g3, g4, g5 and g6) arenormally used for glycosylation. In contrast, the mutation at Asn 104(mutated in gp75g2) did not cause any alteration in molecular massbetween gp75g2 and wild-type gp75; it is most likely that this site isnormally not used for glycosylation.

[0091] To assess the individual roles of each N-glycan in the stabilityand transport of mouse gp75, we performed pulse-chase metabolic labelingwith [³⁵S]methionine followed by immunoprecipitation and Endo Hdigestion on each mutant gp75 transfectant, and compared the data tothat of wild-type mouse gp75 expressed in L cell transfectants. Newlysynthesized wild-type gp75 appeared as a doublet of 70 kDa and 68 kDabands in the transfectants at the end of 15 pulse and after a subsequent15 min, or 30 min chase. Endo H digestion reduced the bands to 57 and 52kDa core polypeptide bands, showing that before 30 min chase, newlysynthesized gp75 remained in the ER. Starting from after 30 min chase,gp75 appeared as a mature 72-79 kDa band, which was resistant to Endo Hdigestion, because Endo H digestion could not remove all N-glycans tothe predicted core peptide size. This indicates movement of gp75 proteinfrom the ER or cis-Golgi (until 30 min chase) to the medial- ortrans-Golgi 30 min after de novo synthesis and further processing on thecarbohydrate chains in these compartment. The 72-79 kDa gp75 protein didnot change further after subsequent chase, indicating completion ofglycosylation on gp75. The mature gp75 is presumably further transportedto the endosomes/lysosomes, although the time course is not reflectedfrom this experiment. The intensity of the 72-79 kDa band remainedstable until 4 h after chase, indicating a half-life of 4-8 h of themature protein.

[0092] The above pulse-chase experiment followed by immunoprecipitationwith mAb TA99 and Endo H digestion reflecting the intracellularstability as well as protein transport from the ER to the Golgi wasperformed on all of the glycosylation mutants. The cellular transportand stability of all the glycosylation mutants can be grouped into 3categories. (1) gp75g1 and gp75g3 appeared to have very similartransport pattern and stability data as that of the wild-type gp75; (2)gp75g4 and gp75g6 proteins remained Endo H sensitive with half-livesbetween 1-4 h, suggesting retention and degradation in the ER; (3)gp75g5 was transported from the ER to the Golgi in a similar rate as thewild type gp75, yet displayed a shorter half life. At the end of 15 minpulse labeling and 30 min chase, gp75g5 appeared as a 68 kDa which wassensitive to Endo H to yield a 57 kDa band. (The additional 66 kDa bandwith a core polypeptide of 52 kDa is a truncated form similar to that infull length transfectants). At the end of 1 h chase, majority of gp75g5was converted to an Endo H resistant 75 kDa band, showing that it wastransported and processed to the medial- or trans-Golgi, and the rate oftransport was similar to that in wild-type transfectants. Unlike that ofthe wild-type gp75, the intensity of the 75 kDa band decreased after 1 hof chase. This suggested that the half life of gp75g5 was between 1 to 4h, shorter than wild type gp75 in transfectants (T1/2=4-8 h).Apparently, the abolishment of the N-linked carbohydrate chain atposition 350 affected the stability of the protein.

[0093] The above data showed that gp75g5 had a shorter half life thanwild-type gp75. It appeared to be transported to the Golgi, andpresumably further to the endosomes/lysosomes, as the transport signalin the cytoplasmic tail is intact. In order to examine whether gp75g5was actually transported to the endosomes/lysosomes and the shorterhalf-life of the protein was due to endosomal/lysosomal degradation, werepeated the pulse-chase experiment in the presence of NH₄Cl, alysosomotrophic weak amine which inhibits proteases in acidicenvironments such as endosomes or lysosomes, or leupeptin, aserine/cysteine protease inhibitor which inhibits mainly proteases inthe lysosomes. At the end of 15 min label, the 68 kDa mutant gp75 bandwas synthesized with equal intensity in the absence or presence of NH₄Clincubation. With the absence of NH₄Cl, the intensity of gp75g5 bandreduced markedly at 4 h chase comparing with that at 0.5 h chase.However, in the presence of NH₄Cl, the gp75g5 band was nearly as strongat the end of 4 h chase as at the end of 0.5 h chase or after 15 minlabeling. This result clearly showed a prolonged half-life to more than4 h for gp75g5 in the presence of NH₄Cl, and suggested that the shorthalf life of gp75g5 was due to rapid degradation in acidic compartmentssensitive to NH₄Cl inhibition, which are most likely the late endosomesor lysosomes. Similarly, in the presence of leupeptin, the intensity ofthe gp75g5 band remained as the same after 4 h chase as after 0.5 hchase, showing a great stabilization of the protein. Based on theseresults, it is concluded that gp75g5 was transported to theendosomes/lysosomes, and was rapidly degraded there.

[0094] The above conclusion is also confirmed by immunofluorescencestaining of transfectants expressing gp75g5 in the absence and presenceof leupeptin. In wild-type gp75 transfectants, gp75 is localized in thejuxtanuclear patches and peripheral punctate vesicles, which representthe Golgi complex and the late endosomes/lysosomes. The juxtanuclearpatches represented Golgi apparatus, and the localization of wild-typefull-length gp75 in the Golgi apparatus at steady state suggestedaccumulation and slow passage of gp75 in this compartment duringtransport. The staining of gp75g5 transfectants showed only intensivejuxtanuclear structures, with no visible peripheral vesicles. The lackof staining of peripheral vesicles indicated that at steady state, therewas no detectable level of gp75g5 in the endosomes and lysosomes.However, staining of gp75g5 transfectants in the presence of leupeptinrevealed an enhanced overall staining of gp75g5 transfectants,particularly, the peripheral vesicles became visible. These peripheralvesicles are most likely endosomes and lysosomes based on studies on thelocation of wild-type gp75 in the transfectants. This result supportsthe above notion that leupeptin stabilized the gp75g5 mutant proteins inendosomes and lysosomes.

[0095] Taken together, the above data showed that the mutation at Asn350 to eliminate an oligosaccharide chain at this position produced amutant gp75 protein, which is more prone to proteolytic digestions inthe lysosomes than the wild-type gp75, and serine or cysteine proteaseswere involved in the degradation process. This mutation did not alterthe route of intracellular sorting and trafficking of gp75, as gp75g5was still sorted to the endosomes/lysosomes.

[0096] Pulse-chase experiments of gp75g1 and gp75g3 showed very similarpattern of gp75 transport and stability compared to the wild-type gp75.This result indicated that the N-glycans at Asn 96 and 181 (eliminatedat gp75g1 and g3) are not involved in determine the stability of gp75.Under immunofluorescence staining, gp75g1 was localized to juxtanuclearstructure and peripheral vesicles, just like the localization ofwild-type gp75. Staining gp75g3 revealed predominantly perinuclearvesicles with non-visible juxtanuclear patches, suggesting localizationof gp75g3 mainly in the endosomes and lysosomes at steady state. Sincethe juxtanuclear patches are most probably the Golgi complex or earlyendosomes, this result suggested an increased rate of transport ofgp75g5 through the Golgi complex and the early endosomes than that ofwild-type gp75. Thus, N-glycan at Asn 181 seems to be involved in therate of transport through the Golgi.

[0097] Pulse-chase experiments of gp75g4 and gp75g6 showed a differentpattern of cellular transport and stability from that of wild-type gp75or other glycosylation mutants. After 15 min pulse and after up to 4 hchase, gp75g4 and gp75g6 mutant proteins remained to be 68 kDa,sensitive to Endo H digestion; and their intensities decreased between 1to 4 h of chase. These data suggested ER retention and degradation ofthe mutant proteins. Under immunofluorescence staining with mAb TA99,gp75g4 showed a pattern of weak, diffuse staining mainly in fineperinuclear networks, indicative of the ER network; while gp75g6 wasmainly localized in condensed perinuclear patches, which was consistentto be the Golgi apparatus. Combining the biochemical and staining data,it appears that gp75g4 is retained in the ER and gp75g6 is retainedmostly in the cis-Golgi apparatus. Thus, the elimination of N-glycan atAsn 304 or Asn 385 affected the cellular transport of the protein fromthe ER to the Golgi. Malfolding may be the mechanism for the retentionas suggested by a lot of earlier studies.

1. A nucleic acid construct for genetic immunization comprising (a) anantigen-coding region encoding an antigenic protein or peptide; and (b)a sorting region encoding a protein or peptide which acts as a sortingsignal to direct intracellular transport of the protein or peptide tothe endosomes or the endoplasmic reticulum of a cell.
 2. The constructof claim 1, further comprising a linker region disposed between theantigen-coding region and the sorting region.
 3. The construct accordingto claim 1 or 2, wherein the sorting region is derived from the humanbrown locus protein, gp75; human albino locus protein, tyrosinase; humansilver locus protein, Pmel 17; or human pink eyed locus P-protein. 4.The construct according to claim 1 or 2, wherein the sorting regionencodes at least the peptide Glu Ala Asn Gln Pro Leu Leu Thr Asp (SEQ IDNO. 1).
 5. The construct according to claim 1 or 2, wherein the sortingregion encodes at least the peptide Glu Glu Lys Gln Pro Leu Leu Met Asp(SEQ ID NO. 2).
 6. The construct according to claim 1 or 2, wherein thesorting region encodes at least the peptide Glu Asp Ser Pro Leu Leu (SEQID NO. 3).
 7. The construct according to claim 1 or 2, wherein thesorting region encodes at least the peptide Glu Asp Thr Pro Leu Leu (SEQID NO. 4).
 8. The construct according to claim 1 or 2, wherein thesorting region encodes at least the peptide sequence Pro Ser Arg Asp ArgSer Arg His Asp Lys Ile His (SEQ ID NO. 5).
 9. The construct accordingto claim 1 or 2, wherein the sorting region is a mutant form in which aglycosylation site present in a corresponding wild type sorting regionhas been altered.
 10. The construct according to any of claims 1 to 9,further comprising a promoter region effective to permit expression ofthe construct in mammalian cells.
 11. The construct according to claim10, wherein the promoter region is selected from among the SV40promoter, the CMV promoter and the RSV promoter.
 12. A vaccine forgenetic immunization comprising a nucleic acid construct according toany of claims 1 to
 11. 13. The vaccine according to claim 12, whereinthe nucleic acid construct is packaged in a liposome.
 14. The vaccineaccording to claim 12, wherein the nucleic acid construct is coated on acolloidal gold particle.
 15. The vaccine according to claim 12, whereinthe nucleic acid construct is incorporated into a viral expressionvector.
 16. A method for inducing an immune response to an antigen in amammal, comprising the step of administering to the mammal a nucleicacid construct or vaccine according to any of claims 1-15.
 17. A methodfor preparing a vaccine for genetic immunization comprising the steppreparing a nucleic acid construct according to any of claims 1 to 11.18. The method according to claim 17, further comprising the step ofpackaging the nucleic acid construct in a liposome carrier.
 19. Themethod according to claim 17, further comprising the step of coating thenucleic acid construct on a colloidal gold particle.
 20. The methodaccording to claim 17, wherein the nucleic acid construct isincorporated into a viral expression vector.